Accurate interpretation of within-host dissemination using barcoded bacteria

Bacterial dissemination across tissues is a critically important process influencing infection outcomes. Monitoring within-host dissemination is challenging because conventional measures of bulk bacterial burden cannot distinguish between lineages that are shared between tissues and those that replicate locally. This limitation can be overcome using barcoded bacteria, where deep sequencing of the barcode locus and comparisons of barcodes between tissues define which lineages spread within the host. Numerous studies have used barcoded bacteria to generate high-resolution maps of dissemination. However, since multiple cells in the infectious inoculum can contain identical barcodes, inferences about dissemination can be confounded when distinct lineages from the inoculum with identical barcodes are observed in different tissues. Thus, even though the same barcodes can be observed in different tissues, dissemination between these tissues may not have occurred. Here, we aimed to develop an approach that would provide a solution to this confounding effect. We developed a simulation-based distance metric that quantifies the significance of observing shared barcodes between tissues. We validated this approach using simulated data sets spanning three orders of magnitude in barcode diversities and on three published experimental infection data sets. Our reanalysis reveals previously unappreciated patterns of <i>Escherichia coli</i> spread during liver abscess formation, clarifies the role of the Muc2 mucin in <i>Listeria monocytogenes</i> systemic spread, and quantifies how <i>Klebsiella pneumoniae</i> replication in the lungs drives systemic dissemination. As barcoding studies expand across diverse infection models, this approach provides an essential tool for accurate interpretation of within-host bacterial dissemination.IMPORTANCEHow microbes move between tissues in the host is an important factor that controls the outcome and severity of infections. A powerful method to monitor within-host microbial dissemination is the use of barcoded bacteria and lineage tracing. Comparisons of barcodes between tissues enable inferences of microbial dissemination, and this method has been applied to diverse contexts of bacterial infections. Here, we demonstrate that inferences of microbial dissemination are confounded, where observing identical barcodes in different tissues does not always signify that dissemination has occurred. To overcome this limitation, we define a metric to quantify the extent to which sharing of barcodes is meaningful and provide new insights into previous barcoding studies in <i>Escherichia coli, Listeria monocytogenes</i>, and <i>Klebsiella pneumoniae</i>. As bacterial lineage tracing continues to be applied across diverse models, our method will help ensure accurate interpretations of microbial dissemination.